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    RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis
    Authors:  Sean D. Gallaher and Melissa S. Roth, date: 04/05/2018, view: 137, Q&A: 0
    Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly ...
    Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
    Authors:  Andrew M. Tidball, Preethi Swaminathan, Louis T. Dang and Jack M. Parent, date: 04/05/2018, view: 166, Q&A: 0
    For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional ...
    Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
    Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types ...
    RNA Purification from the Unicellular Green Alga, Chromochloris zofingiensis
    Authors:  Sean D. Gallaher and Melissa S. Roth, date: 04/05/2018, view: 137, Q&A: 0
    [Abstract] Chromochloris zofingiensis is a unicellular green alga that is an emerging model species for studies in fields such as biofuel production, ketocarotenoid biosynthesis and metabolism. The recent availability of a high-quality genome assembly facilitates systems-level analysis, such as RNA-Seq. However, cells of this alga have a tough cell ...
    Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
    Authors:  Andrew M. Tidball, Preethi Swaminathan, Louis T. Dang and Jack M. Parent, date: 04/05/2018, view: 166, Q&A: 0
    [Abstract] For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector ...
    Isolation of Nuclei in Tagged Cell Types (INTACT), RNA Extraction and Ribosomal RNA Degradation to Prepare Material for RNA-Seq
    [Abstract] Gene expression is dynamically regulated on many levels, including chromatin accessibility and transcription. In order to study these nuclear regulatory events, we describe our method to purify nuclei with Isolation of Nuclei in TAgged Cell Types (INTACT). As nuclear RNA is low in polyadenylated transcripts and conventional pulldown methods would ...
    Detection and Analysis of Circular RNAs by RT-PCR
    Authors:  Amaresh C Panda and Myriam Gorospe, date: 03/20/2018, view: 730, Q&A: 0
    [Abstract] Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory ...
    RNA Cap Methyltransferase Activity Assay
    Authors:  Jackson B. Trotman and Daniel R. Schoenberg, date: 03/20/2018, view: 309, Q&A: 0
    [Abstract] Methyltransferases that methylate the guanine-N7 position of the mRNA 5’ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing ...
    Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
    [Abstract] Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended ...
    CRISPR-mediated Tagging with BirA Allows Proximity Labeling in Toxoplasma gondii
    Authors:  Shaojun Long, Kevin M. Brown and L. David Sibley, date: 03/20/2018, view: 442, Q&A: 0
    [Abstract] Defining protein interaction networks can provide key insights into how protein complexes govern complex biological problems. Here we define a method for proximity based labeling using permissive biotin ligase to define protein networks in the intracellular parasite Toxoplasma gondii. When combined with CRISPR/Cas9 based tagging, this ...
    Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function
    Authors:  Joshua Misa, Cory Schwartz and Ian Wheeldon, date: 03/20/2018, view: 334, Q&A: 0
    [Abstract] The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that ...
    Reduced Representation Bisulfite Sequencing in Maize
    Authors:  Fei-Man Hsu, Chung-Ju Rachel Wang and Pao-Yang Chen, date: 03/20/2018, view: 221, Q&A: 0
    [Abstract] DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 ...
    Microbial Mutagenicity Assay: Ames Test
    [Abstract] The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...